PROJECT SUMMARY Dysbiosis in infants and children is associated with increased susceptibility to inflammatory bowel disease (IBD) in adults. However, the mechanisms whereby the gut microbiota colonization in early life confers health outcomes throughout the lifespan remain unclear. Studies from the previous funding period isolated and cloned a Lactobacillus rhamnosus GG (LGG)-derived secretory protein, p40, and demonstrated that neonatal p40 supplementation prevents colitis in adult mice. Our preliminary studies discovered that p40 interacts with two transcriptional factors, Mga and Max, to regulate expression of Setd1? gene, which encodes a methyltransferase for catalyzing mono and trimethylation of histone 3-lysine 4 (H3K4me1/3). We identified TGF? as a potential target of p40-up-regulated Setd1?. IECs are rapidly renewed and continuously regenerated from intestinal stem cells (ISCs). We found that p40 modulates H3K4m31/3 in ISCs in early stage, and p40 treatment in neonates, but not adult mice, stimulates sustained increase in TGF? gene expression in IECs. TGF? has been shown to have multiple functions against inflammation. Thus, we hypothesize that p40 up-regulates Setd1? gene expression in IECs through increasing Mga:Max dimerization. Supplementation with p40 in early life stimulates Setd1?-dependent H3K4me1/3 deposition at the TGF? locus in ISCs, which is inherited by IECs to enable the sustained increase in TGF? production, and subsequently prevention of intestinal inflammation in adulthood. In Aim 1, we will determine whether p40-stimulated Setd1? gene expression and H3K4me1/3 are required to drive the increase in expressing TGF? gene in IECs, and elucidate whether the interaction between p40 and the Mga:Max dimers mediates up-regulation of Setd1? production. In Aim 2, we will identify the temporal window of p40 exposure in early life that causes the sustained increase in TGF? production in IECs, and define whether p40-regulated sustained increase in TGF? gene expression in IECs requires the increase of Setd1? gene expression inISCs in early life. Human and mouse enteroids and colonoids and 2D cultures and cell lines with silencing Setd1? or Mga genes, and mouse models of constitutive and inducible Setd1? gene knock down in IECs or ISCs will be utilized for these two aims. In Aim 3, we will determine whether the sustained increase in TGF? production after p40 supplementation in early life is required to prevent colitis in adult mice. We will use neutralizing antibodies and inhibitors to block TGF? function in mice with induced and spontaneously developed colitis. We will also determine whether sustained increase in TGF? production enhances Tregs induction in the intestine and protective epithelial responses for the prevention of colitis in adulthood. This aim will be tested in mice with TGF? receptor II deletion in CD4+ T cells and Smad4 deletion in IECs. Together, our proposed research will elucidate a novel mechanism underlying the consequences of long-lasting effects of p40 on prevention of colitis, and lay the foundation for developing early intervention with p40 as a novel therapy for prevention of IBD in adults.